Perspectives of targeted mass spectrometry for protein biomarker verification

The identification of specific biomarkers will improve the early diagnosis of disease, facilitate the development of targeted therapies, and provide an accurate method to monitor treatment response. A major challenge in the process of verifying biomarker candidates in blood plasma is the complexity and high dynamic range of proteins. This article reviews the current, targeted proteomic strategies that are capable of quantifying biomarker candidates at concentration ranges where biomarkers are expected in plasma (i.e. at the ng/ml level). In addition, a workflow is presented that allows the fast and definitive generation of targeted mass spectrometry-based assays for most biomarker candidate proteins. These assays are stored in publicly accessible databases and have the potential to greatly impact the throughput of biomarker verification studies.

Introduction

A biomarker is a measurable indicator that correlates to a specific biological or disease state. Biomarkers play an important role in various clinical applications [1]. Besides screening for an early diagnosis, biomarkers are measured for the classification and staging of diseases in order to assign patients for targeted treatments, to monitor treatment response, and to detect disease recurrence. The process from the discovery of a biomarker to its clinical application can be subdivided into different phases [2, 3]. The initial phase, typically referred to as discovery, aims to produce a list of biomarker candidates through various genomic, transcriptomic, and proteomic technologies. In the following phase, referred to as verification, the correlation of these candidates to the disease is verified over a large cohort of samples. The candidate markers that perform well through the verification are then selected for the clinical validation phase.

Blood plasma is of particular interest as a source for biomarkers, because it is easily accessible and presumably contains quantifiable molecules that provide information characterizing the physiologic and pathologic state of the human body in the form of proteins shed or secreted from the tissue where a pathologic state is present [4, 5]. The major difficulty in finding blood-based biomarkers is the complexity and dynamic range of protein concentrations in human plasma [6, 7]. Tissue-derived proteins, the targets for biomarkers, are found in plasma in the ng/ml concentration range, six orders of magnitude below the classical plasma proteins [6, 8]. In order to identify specific, disease-related, changes in the proteome and circumvent the challenges of plasma proteomics, recent biomarker studies have focused on the analysis of tissue or cell lines for the generation of biomarker candidate lists [8, 9, 10, 11]. This usually leads to a list from hundreds to thousands of candidate proteins, which subsequently need to be verified in human plasma samples. However, despite the large investment and the effort to generate lists of candidates, only a few protein biomarkers are currently used routinely in the clinical setting. In recent years, the rate of newly approved diagnostic markers has been steadily decreasing due in part to the demanding technical requirements for the verification of the candidate proteins in plasma samples [11, 12]. The technology must be sensitive to allow for the quantification of proteins in the ng/ml concentration range in a highly complex background, and all candidates must be quantified with high reproducibility, accuracy, and in a high-throughput manner over large numbers of patient samples.

Currently, the most commonly used approach for verification and clinical validation is the sandwich enzyme-linked immunosorbent assay (ELISA). The advantages of ELISA assays are their high specificity by implementing a pair of antibodies against the candidate protein and their high sensitivity, permitting the quantification of proteins in human plasma at concentrations below the ng/ml range. However, the limiting factors for the ELISA as a technique for plasma biomarker verification are the restricted possibility to multiplex assays and the availability of antibodies for novel candidate proteins, combined with the lengthy and expensive development of new assays. Therefore, the development of an alternate method for protein quantification with high reproducibility and throughput is needed in order to improve the success rate of approved biomarkers [13]. One solution is a targeted quantitative proteomic concept, such as selected reaction monitoring (SRM) (also referred to as multiple reaction monitoring (MRM)) [14^••]. This review focuses on the recent advances in targeted mass spectrometric approaches and their impact on biomarker studies.